This application proposes to devise improved methods for site-directed mutagenesis, and to apply these methods to define regulatory sequences in DNA. We propose to refine oligonucleotide directed mutagenesis, and to explore methods for isolation of mutagenic oligonucleotides from naturally occurring DNAs, as an alternative to synthesis by chemical or enzymatic procedures. We will explore the use of N-hydroxy dCTP as a site-specific mutagen, and will search for other modified nucleotides which are very ambiguously incorporated. As specific applications of these methods we will mutagenize the gene E ribosome binding site of phage PhiX174, and will also mutagenize promoters for human RNA polymerase III occurring on several different sequenced DNAs. We will also map and compare numerous examples of pol III promoters which occur fortuitously on sequenced DNA molecules. These studies could lead to general methods for the precise determination of regulatory sequences in DNA. We propose to use mutations of defined DNA sequence to develop assays for in vivo mutagenesis. Prokaryotic genes carrying mutations of defined sequence will be introduced into mammalian cells in tissue culture. We will attempt to measure mutation rates following reintroduction of the DNA into a bacterial system. As a long-term goal we would like to produce mice-carrying mutant genes of defined sequence whose mutation rates could be measured in E. coli. This could perhaps provide a relatively rapid and economical method for measurement of mutation rates in whole animals.